Dengue virus (DENV) affects more than 100 countries worldwide. Dengue virus infection has been increasing in the southern Peruvian Amazon city of Puerto Maldonado since 2000. We designed this study to describe the prevalence of past DENV infection and to evaluate risk factors. In 2012, we conducted a cross-sectional serosurvey and administered a knowledge, attitudes, and practices (KAP) questionnaire to members of randomly selected households. Sera were screened for antibodies to DENV by enzyme-linked immunosorbent assay and confirmed by plaque reduction neutralization test. We created indices for KAP (KAPi). We used SaTScan (Martin Kulldorff with Information Management Services Inc., Boston, MA) to detect clustering and created a multivariate model introducing the distance of households to potential vector and infection sources. A total of 505 participants from 307 households provided a blood sample and completed a questionnaire. Fifty-four percent of participants (95% confidence interval [CI]: 49.6; 58.5) had neutralizing antibodies to DENV. Higher values of KAPi were positively associated with having DENV antibodies in the multivariate analysis (odds ratio [ORII]: 1.6, 95% CI: 0.6, 2.4; ORIII: 2.7, 95% CI: 1.3, 5.5; and ORIV: 2.4, 95% CI: 1.2, 5.0). Older groups had lower chances of having been exposed to DENV than younger people (OR20-30: 0.5, 95% CI: 0.2, 0.8; OR31-45: 0.5, 95% CI: 0.3, 0.9; and OR>45: 0.6, 95% CI: 0.3, 1.3). Multivariate data analysis from the 270 households with location information showed male gender to have lower risk of past DENV infection (OR: 0.6, 95% CI: 0.4, 0.9). We conclude that risk of DENV infection in Puerto Maldonado is related to gender, age of the population, and location.